Fluorescence

We decided to examine the expression of the two promoters- PrecA and PrecN, and two genes-cheZ and trkD, via the fluorescent intensity of EGFP whose sequence had been ligated downstream after them.

We have constructed a plasmid (pUC18) with EGFP, which is ligated to the plasmid in the PstI and HindIII restriction enzyme cutting sites. Then we link PrecA，PrecN，cheZ，trkD gene to EGFP sequence respectively to construct four plasmids, recA-GFP-pUC18，recN-GFP-pUC18，cheZ-GFP-pUC18 and trkD-GFP-pUC18. We used ultraviolet or nalidixic acid to damage E.coli's DNA to trigger the expression of PrecA and PrecN on in order to examine the expression of EGFP. However, there is a lac promoter upstream before the multi-cloning site of pUC18, which allows us to use IPTG induction to examine the expression of cheZ and trkD by watching the GFP expression. In order to ensure the expression of two genes and GFP, we deleted the terminator of the two genes, cheZ and trkD.

Using the laser scanning confocal microscope (LSCM), we can see obvious fluorescence of cheZ-GFP-pUC18 and trkD-GFP-pUC18(Figure 1 and Figure 2). The fluorescence of trkD-GFP-pUC18, is located mostly on the membrane of E.coli. The lac promoter, however, will express without induction of IPTG, resulting in the expression of GFP of control group, which is cheZ-GFP-pUC18 and trkD-GFP-pUC18 that are not induced by IPTG. Consequently, we utilized the Flow Cytometer(FCM) to exam the intensity of GFP in control group and experiment group. (We are still working on that)

Western Blot test

We planned to exame the expression of gene cheZ and trkD on protein level. Within the inducement of 0.1mg/mL Isopropyl β-D-1-thiogalactopyranoside（IPTG） at 18℃ for 15~18h, we extracted the total proteins of the E.coli transformed with CheZ-pET28a or TrkD-pET32a. Western Blot results showed that the quantity of protein CheZ and TrkD significantly increased after the inducement, indicating that cheZ and trkD expressed well. Additionally, sicne a basal expression caused by promoter lac in both plasmids, the control groups showed a small quantity of proteins.

Cesium absorption test

We use TrkD-pET32a to test the function of trkD. The test could be accomplished through the detection of the amount of Cs+ absorption in the experiment group and control group.

The analysis of Cs+ absorption through trkD indicates that under the inducement of IPTG (0.5mM) for about 4h, the trkD constructed in plasmid can express normally, thus the experiment group has a higher amount of absorption than that of the control group. While the reason why the control has also absorbed Cs+ is that gene trkD also exists in E.coli's genome, so the control group can also express a certain amount of trkD to accomplish the absorption of Cs+. As a result, the absorption of the experiment group is 47.8% higher than that of the control group.

In terms of the result, we have proved that the trkD constructed in the plasmid can function normally.

Characterizing Promoters

Because we want to characterize recA and recN promoters, we turned to Microplate Reader, a machine that can measure the overall fluorescent intensity. We set the exciting wave at 488nm and the emission wave at 520nm, which allow us the measure the fluorescence of recA-GFP-pET28a-BL21 and recA-GFP-pET28a-BL21. After induce with NAL and regulate the OD value of each inocula to a same value, we measured the fluorescence intensity of recA-GFP-pET28a-BL21 and recA-GFP-pET28a-BL21.

About the two crests of the curve of recA-GFP-pET28a-BL21, we guess that the rec promoter inside the genome of the BL21 itself, beside the one caused by rec promoter in the plasmid, may be the reason of another crest. At first, the recA promoter is triggered by the increasing NAL concentration, which leads to the first crest. As the NAL concentration increases, the DNA of the plasmid was damaged but the rec promoter in the genome has not been triggered yet since the concentration of the NAL has not reached its threshold, which results in the trough between the two crests. Finally, the rec promoter in the genome is triggered by the relatively high concentration of NAL and produces the protein of rec family, which may help repair the DNA of the plasmid, which causes the second crest of the curve.

The curve of recN-GFP-pET28a-BL21 seems to support our hypothesis. The curve of recN-GFP-pET28a-BL21 has a platform instead of a trough. Then a crest after that. We guess that the threshold of recN promoter is higher than recA promoter is the reason. The crest caused by the recN promoter in the plasmid overlappes with the trough, which creates a platform as we see.

As the why the the rec promoter of the plasmid will be damaged earlier than that in the genome, we guess the different position of the two promoters is the reason.

However, the difference between all the data is not convincing enough, which may lead to another explanation: all the tendency of the data is caused by system error, and yet, which we haven't found out.